Hydroxyl Radical Overproduction in the Envelope: an Achilles’ Heel in Peptidoglycan Synthesis

While many mechanisms governing bacterial envelope homeostasis have been identified, others remain poorly understood. To decipher these processes, we previously developed an assay in the Gram-negative model Escherichia coli to identify genes involved in maintenance of envelope integrity. One such gene was ElyC, which was shown to be required for envelope integrity and peptidoglycan synthesis at room temperature. ElyC is predicted to be an integral inner membrane protein with a highly conserved domain of unknown function (DUF218). In this study, and stemming from a further characterization of the role of ElyC in maintaining cell envelope integrity, we serendipitously discovered an unappreciated form of oxidative stress in the bacterial envelope. We found that cells lacking ElyC overproduce hydroxyl radicals (HO(•)) in their envelope compartment and that HO(•) overproduction is directly or indirectly responsible for the peptidoglycan synthesis arrest, cell envelope integrity defects, and cell lysis of the ΔelyC mutant. Consistent with these observations, we show that the ΔelyC mutant defect is suppressed during anaerobiosis. HO(•) is known to cause DNA damage but to our knowledge has not been shown to interfere with peptidoglycan synthesis. Thus, our work implicates oxidative stress as an important stressor in the bacterial cell envelope and opens the door to future studies deciphering the mechanisms that render peptidoglycan synthesis sensitive to oxidative stress. IMPORTANCE Oxidative stress is caused by the production and excessive accumulation of oxygen reactive species. In bacterial cells, oxidative stress mediated by hydroxyl radicals is typically associated with DNA damage in the cytoplasm. Here, we reveal the existence of a pathway for oxidative stress in the envelope of Gram-negative bacteria. Stemming from the characterization of a poorly characterized gene, we found that HO(•) overproduction specifically in the envelope compartment causes inhibition of peptidoglycan synthesis and eventually bacterial cell lysis.